PROJECT SUMMARY The liver is the second-most transplanted organ in the United States, with approximately 6,000 transplants per year. Outcomes are limited due to the need for the vast majority of patients to take immunosuppressive drugs for life to prevent allograft rejection. These patients are at increased risk for bacterial or viral infections, cancer, and toxic side effects of the immunosuppressive medications. While several strategies for tolerance induction are effective in rodent models, very few methods have succeeded in inducing tolerance in primates or humans. Transient mixed chimerism, however, achieved kidney allograft tolerance in primates and humans. We developed a nonhuman primate model of allogeneic liver transplantation in order to study the ability of transient mixed chimerism to induce tolerance to liver allografts. We have established the only existing NHP liver allotransplant model in the US, and, to our knowledge, the only active program in the world. Our preliminary data suggest that memory T cell responses (particularly effector/memory CD8 T cells) and the relatively large ischemia-reperfusion inflammatory response (with enhanced levels of IL-6/Th17 cytokines) that results from the operation may represent significant barriers to the induction of tolerance to the liver, despite the development of transient chimerism. Enhanced T cell depletion by targeting host CD2+ memory cells results in enhanced multilineage donor chimerism (including T cell chimerism) and prevents allograft rejection, but led to graft-versus-host disease in one animal due to large numbers of passenger memory T cells in the donor liver. We hypothesize that suppression of both host and donor effector/memory responses by T cell depletion or cytokine modulation will result in donor-specific liver tolerance without graft-versus-host disease. In Aim 1, we will test these hypotheses by comparing the ability of four different immunosuppressive regimens to promote the induction of tolerance to liver transplants in cynomolgus macaques. The first is based on the regimen that successfully achieves tolerance to kidneys in the cynomolgus model and will define the baseline immune response. We will then examine the ability of enhanced donor and recipient T cell depletion with anti- CD2 mAb (regimen 2) +/- modulation of the inflammatory/Th17 response by administering anti-IL-6 receptor mAb (regimen 4) to promote tolerance. We will also perform liver transplants without co-administration of donor bone marrow to define the role of chimerism in modulating the immune response (regimen 3). In Aim 2, we will characterize the cellular, humoral and cytokine response in each regimen, both in the periphery and locally in the graft. The results of these studies will enhance our understanding of the barriers to tolerance induction and the mechanisms of tolerance or rejection in this model. We anticipate that the development of a protocol for safe, rapid and reliable tolerance induction in this pre-clinical NHP model will lead to prompt clinical translation.